Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)lO>a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 pm and thicker than 3 pm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron nicroscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CGSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen filly or pardty engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on twodimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MVF1Oa,
MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or. in siit CLSM. In contrast, the fraction of short MMVF1Oa and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF1Oa, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1.5, and 3.5 times greater, respectively, than those calculated from in sit CLSM d;ata. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results. Key work aerosol, amosite asbestos, ashing, confocal microscopy, fiber burden, man-made fibers, MMVF, respirable, three-dimensional.